Project Summary/Abstract Nitrogenase enzymes perform the conversion of dinitrogen to ammonia and are the lifeblood of the biogeochemical nitrogen cycle. While inherently inert, dinitrogen is made biologically available through these nitrogenase enzymes that ?fix? the gas into a reduced form that subsequently becomes a building block, through amino and nucleic acids, of all forms of life. Nitrogenase enzymes possess multi-metallic core structures within a network of amino acids that are necessary to orchestrate an intricate series of multi-proton and multi-electron transfers. These amino acids provide secondary interactions?though their precise molecular role in substrate capture, substrate activation, and subsequent reduction is not well known. The role of these key secondary interactions in nitrogenase reactivity remains an inherent gap in our knowledge of this vital enzyme. This proposal is targeted at addressing this gap through the design and study of discrete small molecule constructs that contain highly modular and highly directed secondary coordination sphere interactions. This study will extend beyond primary coordination sphere modeling by modifying flexible appended functionalities (boron Lewis acids) of the second coordination sphere to evaluate metal-ligand cooperative small molecule activation. The well-defined secondary sphere interactions will be used to test key mechanistic hypotheses concerning substrate activation/reduction relevant to nitrogenase. These synthetic systems, and the interactions evaluated therein, can, by extension, be adapted to describe biological systems. Our efforts will provide needed contributions to the mechanistic studies of nitrogenase. More broadly, effects of secondary sphere interactions on substrate activation are applicable to many biocatalytic cycles, and these studies can be utilized to illuminate significant contributors to enzymatic reactivity.